Epidermal growth factor is a small polypeptide cytokine that stimulates marked proliferation of epithelial tissues and is a member of a larger family of structurally related cytokines such as transforming growth factor α (TGFα), amphiregulin, betacellulin, heparin-binding EGF and some viral gene products. Abnormal EGF family signalling is a characteristic of certain cancers (Soler, C. & Carpenter, G., 1994 In Nicola, N. (ed) “Guidebook to Cytokines and their Receptors”, Oxford Univ. Press, Oxford, pp 194–197; Walker, F. & Burgess, A. W., 1994, In Nicola, N. (ed) “Guidebook to Cytokines and their Receptors”, Oxford Univ. Press, Oxford, pp 198–201).
The epidermal growth factor receptor (EGFR) is the cell membrane receptor for EGF (Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203–212). The EGFR also binds other ligands that contain amino acid sequences classified as the EGF-like motif. Among these ligands, the three-dimensional structures of EGF and TGFα have been determined by NMR (Montelione, G. T.; Wuthrich, K.; Nice, E. C., Burgess, A. W. and Scheraga, H. A. (1986) PNAS 83(22): 8594–8; Campbell, I. D., Cooke, R. M., Baron, M., Harvey, T. S., and Tappin, M. J. (1989) Prog. Growth Factor Res. 1, 13–22). Upon binding of the ligand to the extracellular domain, the EGFR undergoes dimerization, which eventually leads to the activation of its cytoplasmic protein tyrosine kinase (Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203–212). The EGFR is also known as the ErbB-1 receptor and belongs to the type I family of receptor tyrosine kinases (Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203–212). This group also includes the ErbB-2, ErbB-3 and ErbB-4 receptors. The ligand of ErbB-2 is still unknown but it is clear that heregulin binds to ErbB-3 and ErbB-4 (Plowman, G. D., Green, J. M., Calouscou, J. M., Carlton, G. W., Rothwell, V. M., and Buckley, S. (1993) Nature 366, 473–475). One of the heregulins is known as neuregulin or NDF and contains an EGF-like sequence that was found to fold into an EGF-like fold by NMR (Nagata, K., Kohda, D., Hatanska, H., Ichikawa, S., Matsuda, S., Yamamoto, T., Suzuki, A., and Inagaki, F. (1994) EMBO J. 13, 3517–3523 and Jacobson, N. E., Abadl, N., Sliwkowski, M. X., Reilly, D., Skelton, N. J., and Fairbrother, W. J. (1996) Biochemistry 36, 3402–3417).
The type II family of receptor tyrosine kinases consists of the insulin receptor (INSR), the insulin-like growth factor I receptor (IGF-1), and the insulin receptor-related receptor (Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203–212). Although the type II receptors consist of four chains (α2β2), both the extracellular portions of the receptors from the two families, as well as the tyrosine kinase portions, share significant sequence homology, suggesting a common evolutionary origin (Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203–212, and Bajaj, M., Waterfield, M. D., Schlessinger, J., Taylor, W. R., and Blundell, T. (1987) Biochim. Biophys. Acta 916, 220–226).
The 621 amino acid residues of the extracellular domain of the human EGFR (sEGFR) can be subdivided into four domains as follows: L1, S1, L2 and S2, where L and S stand for “large” and “small” domains, respectively (Bajaj, M., Waterfield, M. D., Schlessinger, J., Taylor, W. R., and Blundell, T. (1987) Biochim. Biophys. Acta 916, 220–226, see FIG. 2). The L1 and L2 domains are homologous, as are the S1 and S2 domains.
Ligand-induced dimerization was first reported for the EGF receptor (Schlessinger, J. (1980) Trends Biochem Sci 13, 443–447) and now is widely accepted as a general mechanism for the transmission of growth stimulatory a signals across the cell membrane. Although many biochemical experiments have been performed to reveal the molecular mechanism of receptor dimerization (Lemnon, M. A., Bu, Z., Ladbury, J. E., Zhou, M., Pinchasi, D., Lax, L., Engelman, D. M., and Schlessinger, J. (1997) EMBO J. 16, 281–294 and Tzabar, E., Pinkas-Kramarski, R., Moyer, J. D., Klapper, D. N., Alroy, L., Levkowitz, G., Shelly, M., Henis, S., Eisenstein, M., Ratzkin, B. J., Sela, M., Andrews, G. C., and Yarden, Y. (1997) EMBO J. 16, 4938–4950 and Lax, L., Mitra, A. K., Ravern, C., Hurwitz, D. R., Rubinstein, M., Ullrich, A., Stroud, R. M., and Schlessinger, J. (1991), J. Biol. Chem. 266, 13828–13833), the molecular mechanism by which monomeric ligands induce dimerization is still unknown for members of the EGFR family. Single particle averaging of electron microscopic images suggests that the overall shape of the sEGFR is four-lobed and doughnut-like (Lax, L., Mitra, A. K., Ravern, C., Hurwitz, D. R., Rubinstein, M., Ullrich, A., Stroud, R. M., and Schlessinger, J. (1991), J. Biol. Chem. 266, 13828–13833). Small angle x-ray scattering also indicates that the sEGFR is a flattened sphere with long diameters of 110 Å and a short diameter of 20 Å (Lemmon, M. A., Bu, Z., Ladbury, J. E., Zhou, M., Pinchasi, D., Lax, L., Engelman, D. M., and Schlessinger, J. (1997) EMBO J. 16, 281–294). The crystallization of sEGFR in complex with EGF has been published (Günther, N., Betzel, C., and Weber, W. (1990) J. Biol. Chem. 265, 22082–22085; Degenhardt M., Weber W., Eschenburg S. Dierks K., Funari S S., Rapp G. and Betzel C. (1998) Acta Crystallogr. D Biol. Crystallogr. 54:999–1001), but the structure has not yet been reported, despite a decade of effort by many groups.
One EGF receptor ligand, TGF-α has been observed to be overproduced in keratinocyte cells which are subject to psoriasis (Turbitt, M. L. et al., 1990, J. Invest. Dermatol. 95(2), 229–232; Higashimyama, M. et al., 1991, J. Dermatol., 18(2), 117–119; Elder, J. T. et al, 1990, 94(1), 19–25). The overproduction of at least one other EGF receptor ligand, amphiregulin, has also been implicated in psoriasis. (Piepkorn, M. 1996, Am. J. Dermatopath., 18(2), 165–171). Molecules that inhibit the EGF receptor have been shown to inhibit the proliferation of both normal keratinocytes (Dvir, A. et al, 1991, J. Cell Biol., 113(4), 857–865) and psoriatic keratinocytes. (Ben-Bassat, H. et al., 1995, Exp. Dermatol., 4(2), 82–88). These findings indicate that EGF receptor antagonists may be useful in the treatment of psoriasis.
Many cancer cells express constitutively active EGFR (Sandgreen, E. P., et al., 1990, Cell, 61:1121–135; Karnes, W. E. J., et al., 1992, Gastroenterology, 102:474–485) or other EGFR family members (Hynes, N. E., 1993, Semin. Cancer Biol. 4:19–26). Elevated levels of activated EGFR occur in bladder, breast, lung and brain tumours (Harris, A. L., et al., 1989, In Furth & Greaves (eds) The Molecular Diagnostics of human cancer. Cold Spring Harbor Lab. Press, CSH, NY, pp 353–357). Antibodies to EGFR can inhibit ligand activation of EGFR (Sato, J. D., et al., 1983 Mol. Biol. Med. 1:511–529) and the growth of many epithelial cell lines (Aboud-Pirak E., et al., 1988, J. Natl Cancer Inst. 85:1327–1331). Patients receiving repeated doses of a humanised chimeric anti-EGFR monoclonal antibody (Mab) showed signs of disease stabilization. The large doses required and the cost of production of humanised Mab is likely to limit the application of this type of therapy. These findings indicate that the development of EGF receptor antagonists will be attractive anticancer agents.